Molecular and Morphological Characterizations of Echinococcus granulosus from Human and Animal Isolates in Kashan, Markazi Province, Iran.

BACKGROUND
One of the most important zoonotic helminths in the world is known as Echinococcus granulosus. Different strains of the E. granulosus have been described based on morphological and molecular characterizations, however, there is limited information regarding the characteristics of the phenotypes and genotypes of E. granulosus in Iran.


METHODS
The present study was prepared to evaluate the phenotypic and genotypic diversity of E. granulosus isolates collected from human, goat, sheep, and cattle based on 19 standard morphometric parameters and mitochondrial and nuclear genes (CO1, ND1, and ITS1) in Kashan, Markazi Province, Iran during 2013-2014.


RESULTS
The biometric analysis for the 19 characters revealed that the 19 morphometric values of cattle isolates were exceptionally higher than human, goat, and sheep isolates (P<0.05). Molecular analysis confirms the morphological findings. Phylogenic analysis of the CO1, NAD1 and ITS1 genes for all isolates, independent of the host, revealed that the common sheep strain (G1) is traveling among livestock in Kashan and the strains are highly adapted to goats, cattle, sheep, and humans.


CONCLUSION
Both morphological and molecular results of this study indicated that the only genotype G1 of E. granulosus travels between humans and other intermediate hosts of this parasite in the area study.


Introduction
ne of the most prevalent, chronic, complicated and an ignored zoonotic disease, caused by the metacestode phase of tapeworm named Echinococcus granulosus is cystic hydatid disease (CHD). The agent of CHD is a worldwide geographical distribution with a great medical and veterinary importance. This tapeworm affects the main organs of humans and domestic animals such as the liver, lungs, and brain (1)(2)(3). CHD has a significant impact on humans as well as slaughtered animal health, with an estimated up to 1.2 million people infected and over 2 billion dollars in annual economic losses from organ condemnation in the slaughter industry (4). In fact, this infection is the leading risk factor for the production of livestock as well as the growing of livestock (5). Poverty, as well as poor sanitation in the raising of livestock, requires essential interventions to eliminate infections and use effective control programs to break the transmission cycle (6). Similarly, in most of the Mediterranean countries and region, hydatidosis is a major public health problem in Iran. Most of the infected areas were located in the northeastern and western of the country, where raising sheep and other livestock farming is common (7). Furthermore, in different regions of Iran, the number of new surgical cases of hydatidosis diagnosed each year is estimated from a nearly 1.18 to 3/ 100000 population (8) and the annual economic loss incurred in all ruminants slaughtered was estimated to be the US $232.2 million (9).
E. granulosus have remarkable forms of phenotypes in that distinguishable based on morphological and biochemical features, but these methods have no capacity for identification, accurate diversity, and interspecies variation of this species (10)(11)(12)(13). Generally, the advent of modern and more sensitive molecular techniques is applied to discriminating a complex intra-specific strain of E. granulosus (12) and showed that the diverse groups of genetic alternatives make up the genotype of E. granulosus (14). Until now, molecular tools such as sequence comparison of mitochondrial DNA are available, which easily identify diversity and DNA polymorphism using suitable genetic markers such as mtDNA (CO1, ND1) genes in both intermediate and definitive hosts (15)(16)(17). This approach could help clarify the epidemiological situation and suit control programs of disease in a certain area (3). Analyses of mitochondrial and nuclear genomes have been in an extensive range to categorize strains of E. granulosus into distinct genotypes. To date based on phenotypic characters and analysis of gene sequences, a high intraspecific diversity, as well as a complex consist of at least 10 (G1-G10) distinct strains or genotypes. Five species of E. granulosus have been reported worldwide and have introduced the new molecular phylogeny and taxonomy of the parasite (18,19). These species and genotypes are E. granulosus sensu stricto (G1-3, sheep/buffalo strains), E. equinus (G4, horse strain), E. ortleppi (G5, cattle strain), E. canadenrsis (G6-7, camel-pig strain, G8, American cervid strain, G10 Fennoscandian cervid strain) (15,18,(20)(21)(22)(23). Recently molecular studies described a new specific genotype, named E. felidis found in lions (24).
This genetic characterization and the extensive different variants have been very notable in better comprehension and understanding of the life cycle pattern (25,26) as well as other biological features of E. granulosus, such as sensitivity to chemotherapy and pathologic patterns (27,28). The complex of G1-3 is the most major genotype that is responsible for human infection for most of the global burden. Among these variants, the most common are G1 (sheep strain) (29), while limited events have been attributed to the camel strain (30,31). Other genotypes are rare pathogenesis of human disease. The G4 genotype (horse stain) O have not affected human (12). The phylogeny for E. granulosus was reconstructed due to the structures of the entire mitochondrial genome (22) as well as several nuclear markers (32,33). Iran is one of the most widespread areas of unilocular hydatid cyst disease where numerous classes of intermediate hosts are frequently infected with E. granulosus. Currently, numerous molecular studies demonstrated several genotypes of E. granulosus, including complex G1-3 (sheep/buffalo strains) and G6 (camel strain) have been genotypically recognized from sheep, cattle, camel, as well as humans in different parts of Iran. These investigations clearly showed that the G1 genotype of E. granulosus is the major cause of hydatidosis in various regions of the country (11,17,(34)(35)(36)(37)(38)(39)(40)(41)(42)(43).
Identification of E. granulosus strains has transmitted in countless regions using diverse methods, including morphology and molecular genetics, which all of these methods have demonstrated that they are beneficial when used to accompany. Furthermore, concurrent use of phenotypic and genotypic characterization could prove more precise and dependable information regarding the nature of diversity within E. granulosus strains (44). For the design of control strategies and clarifying epidemiology and taxonomy of parasite in an endemic area, identification of E. granulosus variants using molecular and genetic methods is very important (21,45,46). Detection of genetic diversity of the larval stage and adult worms of E. granulosus for accurate understanding patterns, the transmission in endemic regions of Iran is important, where more than one species of intermediate host is present (43,47,48).
The aim of the present study was to identify, both genetically as well as morphologically characteristics of E. granulosus isolates from humans, as well as different domestic animals (sheep, goats and cattle) from Kashan region, Isfahan Province, Iran using the partial sequence of rDNA (ITS1) and mtDNA (CO1 and ND1) genes. Such knowledge should allow a better comprehension of the molecular epidemiology and control of hydatidosis, as well as determining the nature and extent strains of Echinococcus in various regions of Iran.

Sampling design
During the period Apr 2013-Feb 2014, internal organs with unilocular hydatid cyst were composed of 15 patients who underwent surgery at the Shahid Beheshti Hospital and 33 sheep, 20 cattle, as well as 32 goats slaughtered at abattoirs in Kashan City, Isfahan Province, Iran. All of the patients that suffered from cystic echinococcosis were identified by histopathological examination (PAS staining) and imaging of the respective tissues. Samples of hydatid fluid aspirated from each hydatid cyst were collected, transported into a single tube and washed 3 times with sterile PBS (pH 7.2). Before fixing samples in 70% ethanol, the fluid cysts were controlled for the occurrence of protoscolices. The possibility of protoscolices was checked microscopically by using 0.1% aqueous eosin stain. All specimens were stored in 4 °C for fu ture analysis.
Approval to this study was granted by Kashan University of Medical Sciences Ethical Committee (document 2731).

Morphological analysis
Four hundred protoscolices from 100 cysts were isolated from liver and lung of human, sheep, cattle and goats and were analyzed for morphological characterization. The isolated protoscolices from each infected host were recognized by the diagnostic keys (49). Measurements of character were made on both small as well as large hooks per rostellum from all of the 400 protoscolices for each isolate. The total length (TL), total number of hooks (NH), blade length (BL), total width (TW), blade width (BW), handle width (HW), handle length (HL), as well as the distance between the blade and guard (BGD), were calculated by a calibrated ocular micrometer at magnifications of ×100 and ×400. The isolated protoscolices were transmitted into an individual plate, washed 3 times with PBS, and saved in 70% ethanol until mole cular works.
The morphological differences between all studied hosts were analyzed with ANOVA and Student's t-test using SPSS ver. 16 (SPSS Inc., Chicago, IL, USA).

Molecular characterization Genomic DNA extraction
For the genomic DNA extraction, the protoscolices were thoroughly rinsed numerous times with sterile phosphate buffer solution (PBS) (pH 7.2) to eliminate the ethanol prior to DNA extraction (50). Genomic DNA was extracted using Kit (Bioneer; Korea), according to manufacturer's instruction with brief modifications. Roughly, 2 ml packed volume of protoscolices were mechanically ground in 200µl lysis buffer and 30µl proteinase K and incubated at 55 °C for 3 min. The purified DNA was eluted to a final volume of 30-50 µl in elution buffer (EL) and stored at -20 o C until molecular analysis. The concentration and quality of the DNA were determined using both spectrophotometric and gel electrophoresis methods.

PCR-RFLP analysis
Fragments of ITS1, ND1, and CO1 genes were amplified from each isolates using primer pairs, previously described (15,17). PCRs containing between 100 ng of DNA was performed using the following conditions: 1.  (51). The PCR products, with a 100 bp DNA ladder, were separated by electrophoresis on 1.5% agarose gels, observed, and analyzed under UV transillumination. E. granulosus DNA samples were evaluated by PCR-RFLP of the genes coding for ND1, CO1, and ITS1, as previously described (Bowles and McManus, 1993) with slight modification. All PCR products were digested using restriction enzymes, including HaeIII, RsaI, HpaII for ITS1, ND1, and COX1 respectively, using buffer recommended by the manufacturer (Thermo Scientific) in a final 20µl volume. The restriction fragments were separated by electrophoresis on 3.5% agarose gel, and stained with ethidium bromide and visualized under UV transillumination.

Genomic DNA sequencing and phylogenetic analysis
Phylogenetic assay was determined by analyzing genomic (rDNA, ITS1) and mitochondrial DNA (mtDNA ND1 and CO1). For this propose, 27 amplicons, representing each unique RFLP profile, were selected. Furthermore, the different RFLP-PCR products were purified using the AccuPrep® Gel Purification Kit (Bioneer; Korea) according to the manufacturer's guidelines. The concentration of DNA was estimated by comparison with a DNA Marker (100bp) in 3.5% agarose gel. All resulting PCR products were sequenced by targeting genes (ND1, COX1, and ITS1) in both directions using the said primers by the ABIPRISMTM 3130 Genetic Analyzer automated sequencer (Applied Biosystems, USA). All sequences were compared with sequences of E. granulosus available in GenBank sequences of all regional species using the Chromas software (version 3.1). The nucleotide sequence analysis was done using the BLAST algorithms from the National Center for Biotechnology. Phylogenetic trees and progression analyses were constructed using Tamura 3-parameter option of the neighbor-joining model with MEGA6 software (52). Taenia multicepes (JX535576) were used as an out-group. The bootstrap scores were calculated for 2000 replicates.

Morphological analysis
The results of the morphometric characterizations 400 protoscolices of E. granulosus from Kashan, Iran are presented in Table 1.
The arrangement of small and large hooks in humans, sheep, goat, and cattle isolates were similar. The results clearly showed a high degree of phenotypic variation between protos-colices of human, sheep, goat and cattle isolates in Kashan area.
According to the data, all 19 morphometric values detected from large and small hooks demonstrated of cattle isolates have significantly higher isolates than humans, sheep, and goats, other than the additional morphometric values of cattle isolate, which was suggestively higher than the other (P<0.05).

Molecular analysis
The obtained samples were related to cysts of the liver (55%) and lung (45%). As predicted, PCR products size of a 1000 bp fragment of the ITS1 rDNA as well as 830 bp and 444 bp fragments of the ND1 and the CO1 mtDNA respectively were provided from all parasite samples (Fig. 1). All of the E. granulosus isolates were tested by the PCR-RFLP evaluation of the ITS1, ND1, and CO1 using restriction endonucleas-es (HaeIII, RsaI, and HpaII). The RFLP patterns of all cattle, sheep, goat, as well as human isolates, were identical ( Fig. 2-4).  gene. The phylogenetic evaluation of the sequence data showed no host specificity among genotype. The phylogenetic analysis of concatenated sequences of CO1, ND1, and ITS1 was showed on the cluster, and represent all strains related to G1 genotype (Fig. 5).

Discussion
Cystic echinococcosis is one of the essential public health complications in most of the Mediterranean region ' s countries, including Iran, where the highest occurrence of disease in humans, as well as other intermediate hosts, is found (47,48). Huge economic burdens and lack of resources are significant causes of the increasing of diseases as a public health problem (53).
In recent years, many investigations were undertaken regarding the importance of extending genotypic and phenotypic diversity in the dog tapeworm E. granulosus, the source of hydatid cyst disease (21). These variations have significant outcomes for the evolution of diagnostic techniques of E. granulosus. Molecular discrimination within and between E. granulosus detected by application of molecular methods triggered to divide this parasite to 10 distinct genotypes as each one occurs to have individual phenotypes characteristic (18). E. granulosus have 10 define genotypes (G1-10) in different regions of the world (18,21). The most frequent and global public health problem associated with human disease is the common sheep strain (G1) distributed in all countries (5,8,18,27,28,54). The design and control strategies are contingent upon such evidence and, in particular, mode of transmission is a threat to human health. E. granulosus, as a species, has extensive phenotypic and genotypic variation, which vary in morphology, life cycle patterns, host specificity, as well as other states (14). For identification strains/genotypes of E. granulosus, according to extensive research, different methods have been carried out, and each of them have sensitivity and accuracy for detection of Iranian genotypes, however, using both morphological and molecular analytic methods could afford scientific and relevant knowledge around the extent and significance of dissimilarity within E. granulosus, the causative agent of hydatid disease (35,54).
The present study showed a high level of morphological variations in large and small rostellar hook metacestodes of E. granulosus from cattle, sheep, goats, and humans. In spite of, biochemical analysis can prepare beneficial information on the recognition of genotypes of E. granulosus from various hosts, this approach can be undecided and limited about identify strains (14). Although morphological criteria for discrimination of E. granulosus may be doubtful, it is a quick, valid, and economical method for identifying E. granulosus strains in Iran, as well as a good tool for epidemiological studies (10,55). Therefore, using both as molecular as well as morphological genetics methods simultaneously could provide more careful and dependable evidence regarding the nature and extent of variation within E. granulosus isolate (21).
In the present study, all 19 morphometric indices drastically among domestic animals (sheep, goat, and cattle) as well as human isolates obtained from the area study. Therefore, morphometric evaluation was discovered to be an advantageous tool for variance identifi-cations of common strains of E. granulosus from Iran. Data acquired from the morphological analysis in the present study are comparable to those conveyed by other investigators of Iran (10,35,56). Analysis of the rostellar hooks of E. granolusus declared that in transmission of the larval stage to definitive hosts, characteristics of the hook may be stayed stable with less variation than adult worm. This could identify the strain of this tapeworm and the source of infection (49,57). The total length of the large hook and the blade of large hook characters of Iranian animals were significantly different from Egyptian ones (40). These dissimilarities insinuate that there is possibly an inter-group heterogeneity among Egyptian as well as Iranian isolates. At present, various techniques have focused on the molecular identification of E. granulosus, while an increasing number of them used research on the parasites nuclear genes as well as mitochondrial NADH dehydrogenase 1(ND1) and cytochrome oxidase 1(CO1) (41)(42). Recently in Iran, several molecular studies showed the diversity of mitochondrial and ribosomal genes and confirmed 3 distinct E. granulosus genotypes, including genotype G1 in cattle, sheep, camels, goats, as well as humans, genotype G3 in sheep, humans, buffalo, as well as cattle, and genotype G6 in humans, sheep, as well as camels in different geographic areas (12,20,26,(43)(44)(45)(46). Camels, as well as sheep strains, are comparable identified in humans as well as cattle.
The results of this study showed that G1 genotype (sheep strain) was the predominant genotype of E. granulosus in infected humans as well as domestic animals, which is in agreement with other reports from Iran and other countries (17,34,36,40,48). The usage of mitochondrial DNA sequencing is primarily centered on its rapid evolution, emphasizing its significance in the biasness of closely affiliated organism as in the event of Echinococcus genotypes, which appear to be favorably homogenous evolutionary units (58).

Conclusion
This study, for the first time based on morphometric and molecular-phylogenetic taxonomic, indicated that along strain (G1 sheep strain) of E. granulosus distributes amongst the intermediate masses of this parasite Kashan, Iran. Therefore, similar to additional transmittable diseases, hydatidosis is essential in being regarded as an important concern in the health policy makers' decisions. Our genetic characterization of human and animal E. granulosus strain in Iran will be quite useful regarding determination strains of taxonomy and development prevention strategies as well as control programs of infected hosts, particularly when there is a shortage of evidence regarding the character of this worm that affects intermediate and definitive hosts. Further studies including additional sample sizes from diverse geographic regions of Iran are necessary for genetic mapping of E. granulosus.